Methods for monitoring cell proliferation, differentiation, and function using flow cytometry have enabled investigation of complex biological phenomena, e.g., immune responses to antigen, which responses involve complex interactions among multiple cell types (see, Wallace P K, Trio J D, Jr., Fisher J L, Wallace S S, Ernstoff M S, Muirhead K A. Tracking Antigen-Driven Responses by Flow Cytometry: Monitoring Proliferation by Dye Dilution. Cytometry Part A 73A: 10194034, 2008). So called cell-tracking dyes have proven useful for qualitative and quantitative monitoring of cell division, both in vivo and in vitro (see, Hawkins E D, Hommel M, Turner M L, Battye F L, Markham J F, Hodgkin P D. Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat Protoc 2007, 2:2057-2067; and Wallace P K, Muirhead K A. Cell tracking 2007: A proliferation of probes and applications. Immunol Invest 2007, 36:527-561). These dyes, also referred to herein as cell-tracking reagents which term is inclusive of cell-tracing reagents, generate a fluorescent signal that, while relatively stable in non-dividing cells, progressively decreases with each round of cell division. Reduction in fluorescence intensity can be quantified by flow cytometry in conjunction with any of several different algorithms to estimate the extent of proliferation (in response to a particular stimulus) based on dye dilution (see, Wallace et al., Cytometry Part A 73A: 1019-1034, 2008). A major advantage of using flow cytometry in conjunction with cell-tracking reagents to monitor the extent of cell division is that cells can also be stained for expression of other cell surface or intracellular markers to define lineage, functionality, activation state, cytokine expression, etc. (see, Bercovici N, Givan A L, Waugh M G, Fisher J L, Vernel-Pauillac F, Ernstoff M S, Abastado J P, Wallace P K. Multiparameter precursor analysis of T-cell responses to antigen, J Immunol Methods 2003, 276:5-17; Fazekas de St Groth B, Smith A L, Koh W P, Girgis L, Cook M C, Bertolino P. Carboxyfluorescein diacetate succinimidyl ester and the virgin lymphocyte: A marriage made in heaven. Immunol Cell Biol. 1999, 77:530-538; and Tanaka Y, Ohdan H. Onoe T, Asahara T. Multiparameter flow cytometric approach for simultaneous evaluation of proliferation and cytokine-secreting activity in T cells responding to alto-stimulation. Immunol Invest 2004, 33:309-324).
Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, or, alternatively, CFSE) remains a popular, commercially available cell-tracking reagent, excitable with 488-nm laser light to give a bright green fluorescence. In combination with another cell-tracking reagent, i.e., the 647 nm-excitable red-fluorescent lipophilic membrane dye PKH26, CFDA-SE has been widely used to monitor cell proliferation by flow cytometry in heterogeneous cell populations. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division.
Notwithstanding the current popularity of CFDA-SE, there remains a need for alternative fluorescent dyes, useful as cell-tracking reagents, with different spectral properties. Such reagents may be combined for simultaneous use not only with CFDA-SE, but also with other currently-available cell-tracking reagents, such as, for example, the 488 nm-excitable reagent Green Fluorescent Protein (GFP), or with the 647 nm-excitable red-emitting dye PKH26. Such use thereby permitting researchers to study cell proliferation, differentiation, and/or function in otherwise indistinguishable cell populations in mixed cell cultures with multi-color applications using flow cytometry.
Many 7-hydroxycoumarin derivatives have been widely used for labeling biological molecules, e.g., proteins inside cells, due to their favorable fluorescent properties. Sun et al. developed a series of fluorinated 7-hydroxycoumarin analogs and demonstrated them to be highly fluorescent and photostabile (see, Sun W-C, Gee K R, Haugland R P. Synthesis of novel fluorinated coumarins: Excellent UV-light excitable fluorescent dyes. Bioorg and Med Chem Letters, 1988, 8, 3107-3110). Such analogs have been used for the preparation of fluorescent protein conjugates as well as substrates for a variety of enzymes, including, but not limited to, phosphatases, β-lactamases, and β-galactosidases. In particular, 3-carboxy-6,8-difluoro-7-hydroxycoumarin (otherwise known as Pacific Blue™ dye) has been broadly employed as a 405 nm violet laser-excitable protein labeling reagent (see, Gee K R, Haugland R P, Sun W-C. Derivatives of 6,8-difluoro-7-hydroxycoumarins. U.S. Pat. No. 5,380,912 (issued Nov. 3, 1998)). More recently and in conjunction with flow cytometry, Abrams et al. described 3-carboxy-6-chloro-7-hydoxycoumarin as a violet laser-excitable protein labeling reagent having a brightness similar to that of Pacific Blue™ dye (see, Abrams B, Diwu Z. Guryev O, Aleshkov S, Hingorani R, Edinger M, Lee R, Link J, Dubrovsky T. 3-Carboxy-6-chloro-7-hydroxycoumarin: A highly fluorescent, water-soluble violet-excitable dye for cell analysis. Anal Biochem, 2009, 386, 262-269). However, neither the aforementioned Sun et al. nor Abrams et al. references describe compounds applicable for use as cell-tracking reagents in long-term studies of cell proliferation, differentiation, and/or function.
Recently, CellVue® Lavender has been employed as a new, cell-tracking reagent utilizing 405 nm violet laser light excitation. However, CellVue® Lavender appears to suffer from limitations including, for example, low fluorescence intensity and a broad emission wavelength well above 550 nm, the combination of which makes its use non-ideal in multi-color applications using flow cytometry.
The development of cell-tracking reagents efficiently excitable with a 405 nm violet laser to provide bright fluorescence intensity for long-term monitoring of cell proliferation, differentiation, migration, location, and/or function using flow cytometry has, heretofore, not been realized.